The present invention relates generally to integrin mediated diseases and, more particularly, to nucleic acids encoding xcex1vxcex23-inhibitory monoclonal antibodies and to CDR grafted xcex1vxcex23-inhibitory antibodies for the therapeutic treatment of xcex1vxcex23-mediated diseases.
Integrins are a class of cell adhesion receptors that mediate both cell-cell and cell-extracellular matrix adhesion events. Integrins consist of heterodimeric polypeptides where a single xcex1 chain polypeptide noncovalently associates with a single xcex2 chain. There are now about 14 distinct xcex1 chain polypeptides and at least about 8 different xcex2 chain polypeptides which constitute the integrin family of cell adhesion receptors. In general, different binding specificities and tissue distributions are derived from unique combinations of the xcex1 and xcex2 chain polypeptides or integtin subunits. The family to which a particular integrin is associated with is usually characterized by the xcex2 subunit. However, the ligand binding activity of the integrin is largely influenced by the xcex1 subunit. For example, vitronectin binding integrins contain the xcex1v integrin subunit.
It is now known that the vitronectin binding integrins consist of at least three different xcex1v containing integrins. These xcex1v containing integrins include xcex1vxcex23, xcex1vxcex21 and xcex1vxcex2h5, all of which exhibit different ligand binding specificities. For example, in addition to vitronectin, xcex1vxcex23 binds to a large variety of extracellular matrix proteins including fibronectin, fibrinogen, laminin, thrombospondin, von Willebrand factor, collagen, osteopontin and bone sialoprotein I. The integrin xcex1vxcex21 binds to fibronectin, osteopontin and vitronectin whereas xcex1vxcex25 is known to bind to vitronectin and osteopontin.
As cell adhesion receptors, integrins are involved in a variety of physiological processes including, for example, cell attachment, cell migration and cell proliferation. Different integrins play different roles in each of these biological processes and the inappropriate regulation of their function or activity can lead to various pathological conditions. For example, inappropriate endothelial cell proliferation during neovascularization of a tumor has been found to be mediated by cells expressing vitronectin binding integrins. In this regard, the inhibition of the vitronectin-binding integrin xcex1vxcex23 also inhibits this process of tumor neovascularization. By this same criteria, xcex1vxcex23 has also been shown to mediate the abnormal cell proliferation associated with restenosis and granulation tissue development in cutaneous wounds, for example. Additional diseases or pathological states mediated or influenced by xcex1vxcex23 include, for example, metastasis, osteoporosis, age-related macular degeneration and diabetic retinopathy, and inflammatory diseases such as rheumatoid arthritis and psoriasis. Thus, agents which can specifically inhibit vitronectin-binding integrins would be valuable for the therapeutic treatment of diseases.
Many integrins mediate their cell adhesive functions by recognizing the tripeptide sequence Arg-Gly-Asp (RGD) found within a large number of extracellular matrix proteins. A variety of approaches have attempted to model agents after this sequence to target a particular integrin-mediated pathology. Such approaches include, for example, the use of RGD-containing peptides and peptide analogues which rely on specificity to be conferred by the sequences flanking the RGD core tripeptide sequence. Although there has been some limited success, most RGD-based inhibitors have been shown to be, at most, selective for the targeted integrin and therefore exhibit some cross-reactivity to other non-targeted integrins. Such cross-reactive inhibitors therefore lack the specificity required for use as an efficacious therapeutic. This is particularly true for previously identified inhibitors of the integrin xcex1vxcex23.
Monoclonal antibodies on the other hand exhibit the specificity required to be used as an effective therapeutic. Antibodies also have the advantage in that they can be routinely generated against essentially any desired antigen. Moreover, with the development of combinatorial libraries, antibodies can now be produced faster and more efficiently than by previously used methods within the art. The use of combinatorial methodology also allows for the selection of the desired antibody along with the simultaneous isolation of the encoding heavy and light chain nucleic acids. Thus, further modification can be performed to the combinatorial antibody without the incorporation of an additional cloning step.
Regardless of the potential advantages associated with the use of monoclonal antibodies as therapeutics, these molecules nevertheless have the drawback in that they are almost exclusively derived from non-human mammalian organisms. Therefore, their use as therapeutics is limited by the fact that they will normally elicit a host immune response. Methods for substituting the antigen binding site or complementarity determining regions (CDRs) of the non-human antibody into a human framework have been described. Such methods vary in terms of which amino acid residues should be substituted as the CDR as well as which framework residues should be changed to maintain binding specificity. In this regard, it is understood that proper orientation of the xcex2 sheet architecture, correct packing of the heavy and light chain interface and appropriate conformation of the CDRs are all important for preserving antigen specificity and affinity within the grafted antibody. However, all of these methods require knowledge of the nucleotide and amino acid sequence of the non-human antibody and the availability of an appropriately modeled human framework.
Thus, there exists a need for the availability of nucleic acids encoding integrin inhibitory antibodies which can be used as compatible therapeutics in humans. For xcex1vxcex23-mediated diseases, the present invention satisfies this need and provides related advantages as well.
The invention provides a Vitaxin antibody and a LM609 grafted antibody exhibiting selective binding affinity to xcex1vxcex23. The Vitaxin antibody consists of at least one Vitaxin heavy chain polypeptide and at least one Vitaxin light chain polypeptide or functional fragments thereof. Also provided are the Vitaxin heavy and light chain polypeptides and functional fragments. The LM609 grafted antibody consists of at least one LM609 CDR grafted heavy chain polypeptide and at least one LM609 CDR grafted light chain polypeptide or functional fragment thereof. The invention additionally provides a high affinity LM609 grafted antibody comprising one or more CDRs having at least one amino acid substitution, where the xcex1vxcex23 binding activity of the high affinity LM609 grafted antibody is enhanced. Nucleic acids encoding Vitaxin and LM609 grafted heavy and light chains as well as nucleic acids encoding the parental non-human antibody LM609 are additionally provided. Functional fragments of such encoding nucleic acids are similarly provided. The invention also provides a method of inhibiting a function of xcex1vxcex23. The method consists of contacting xcex1vxcex23 with Vitaxin or a LM609 grafted antibody or functional fragments thereof under conditions which allow binding to xcex1vxcex23. Finally, the invention provides for a method of treating an xcex1vxcex23-mediated disease. The method consists of administering an effective amount of Vitaxin or a LM609 grafted antibody or functional fragment thereof under conditions which allow binding to xcex1vxcex23.